Abstract Normal lymphocyte development relies on a series of cues from cell surface receptors. This includes receptors for important cytokines, such as IL-7 and receptors (pre-BCR and pre-TCR) generated after successful assembly of antigen receptor genes. We have demonstrated that the activation of DNA damage responses (DDR) by DNA double strand breaks (DSBs) generated by RAG during V(D)J recombination also provides important cues for developing lymphocytes. Like all DSBs, RAG DSBs activate the ATM kinase, which coordinates canonical DDR including repair by non-homologous end joining (NHEJ), initiation of cell cycle arrest and activation of cell death if the DSBs persist unrepaired. However, we demonstrated that in response to RAG DSBs ATM also activates a genetic program that includes many genes encoding proteins that have no function in canonical DDR. Rather these proteins function in lymphocyte-specific processes that could be important for normal development. From this we proposed that RAG DSBs, and possibly other types of physiologic DSBs, provide important signals that regulate cell-type-specific processes. In the last period of this grant we focused on understanding how RAG DSB signals activate distinct transcription pathways and how the resulting genetic program influences B cell development. In this regard, we have shown that the activation of ATM by RAG DSBs generated during immunoglobulin light (Igl) chain gene assembly in pre-B cells leads to the induction of both NF-kB1 and NF-kB2. NF-kB2 up-regulates the expression of the SpiC transcriptional repressor, which inhibits pre-BCR signaling by down-regulating the expression of Syk and BLNK. We find that while NF-kB1 can be activated by both RAG DSBs and genotoxic DSBs (from ionizing radiation), NF-kB2 is only activated by RAG DSBs. This is a very important finding as it demonstrates that the DSBs generated during antigen receptor gene assembly can specifically activate some transcriptional pathways that are not generally activated by all DSBs. In the current proposal we will establish the mechanistic basis for the selective activation of NF-kB2 by RAG DSBs. This will provide an important paradigm for our understanding of the activation of tissue-specific responses by different types of physiologic DSBs. Moreover, our finding that RAG DSB signals inhibit pre-BCR signals has led us to propose a ?toggle? model for pre-BCR and DDR signaling in pre-B cells. We envision that pre-B cells toggle between pre-BCR signals that promote Igl chain gene assembly and the activation of DDR by the resulting RAG DSBs that inhibit pre-BCR signaling and thus additional Igl chain gene rearrangements. This toggling between pre-BCR and DDR signals would provide for ordering of Igl chain gene assembly and promote genome stability in developing B cells.